Margioris A N, Markogiannakis E, Makrigiannakis A, Gravanis A
Department of Clinical Chemistry, University of Crete School of Medicine, Iraklion, Greece.
Endocrinology. 1992 Aug;131(2):703-9. doi: 10.1210/endo.131.2.1353443.
The PC12 is a cloned rat pheochromocytoma cell line that retains a number of chromaffin cell characteristics, such as the presence of nicotinic cholinergic receptors, the synthesis and secretion of catecholamines, and the expression of a number of neuropeptide genes. The PC12 cell line is a useful model for the study of neuronal development, since PC12 cells can be induced to differentiate toward sympathetic neurons after exposure to nerve growth factor (NGF). PC12 cells can also be induced to differentiate toward the opposite direction, i.e. toward mature chromaffin cells. Morphological and biochemical changes mark the differentiation of PC12 cells toward either direction. Among the substances proposed as biochemical markers of PC12 cell differentiation toward chromaffin cells is the endogenous opioid precursor proenkephalin and its posttranslational peptide products. Indeed, the proenkephalin gene is expressed in both adrenal chromaffin and PC12 cells. The secretion of enkephalins from PC12 cells increases by several-fold after differentiation toward chromaffin cells. On the other hand, prodynorphin (another endogenous opioid precursor) is not present in normal adrenal chromaffin cells, but it is synthesized by human pheochromocytomas. It, thus, appears that dedifferentiation of chromaffin cells induces expression of the prodynorphin gene. Consequently, the aim of this study was to determine whether the rat pheochromocytoma-derived PC12 cell line expresses the prodynorphin gene, if it secretes dynorphins, and if the NGF-induced differentiation of PC12 cells toward sympathetic neurons affects the secretion of the latter. We found the following. 1) PC12 cells synthesize prodynorphin and secrete its peptide products. 2) The size of the prodynorphin transcript and the mol wt of its dominant form of dynorphin appear to be similar or identical to those of prodynorphin in rat anterior pituitary. 3) PC12 dynorphin secretion is increased, in a dose-dependent manner, after nicotinic cholinergic stimulation, an effect blocked by the specific nicotine antagonist hexamethonium. Thus, it appears that after cholinergic stimulation, PC12 dynorphin is cosecreted with catecholamines, a phenomenon described for a number of neuropeptides, including the proenkephalin-derived opioids. 4) The NGF-mediated differentiation of PC12 cells into sympathetic neurons exerted a stimulatory effect on basal, nicotine-induced, and depolarization-dependent dynorphin secretion. However, NGF did not shift the nicotine dose-response curve of dynorphin secretion. In conclusion, it appears that while changes in the secretion of proenkephalin-derived peptides may serve as a marker of PC12 cell differentiation toward chromaffin cells, an increase in the secretion of prodynorphin-derived peptides may represent a marker of NGF-induced differentiation of PC12 cells toward sympathetic neurons.
PC12是一种克隆的大鼠嗜铬细胞瘤细胞系，保留了许多嗜铬细胞的特征，如存在烟碱型胆碱能受体、儿茶酚胺的合成与分泌以及多种神经肽基因的表达。PC12细胞系是研究神经元发育的有用模型，因为PC12细胞在暴露于神经生长因子（NGF）后可被诱导分化为交感神经元。PC12细胞也可被诱导向相反方向分化，即向成熟嗜铬细胞分化。形态学和生化变化标志着PC12细胞向任一方向的分化。在被提议作为PC12细胞向嗜铬细胞分化的生化标志物的物质中，有内源性阿片肽前体脑啡肽原及其翻译后肽产物。事实上，脑啡肽原基因在肾上腺嗜铬细胞和PC12细胞中均有表达。PC12细胞向嗜铬细胞分化后，脑啡肽的分泌增加了几倍。另一方面，强啡肽原（另一种内源性阿片肽前体）在正常肾上腺嗜铬细胞中不存在，但由人嗜铬细胞瘤合成。因此，似乎嗜铬细胞的去分化诱导了强啡肽原基因的表达。因此，本研究的目的是确定源自大鼠嗜铬细胞瘤的PC12细胞系是否表达强啡肽原基因，是否分泌强啡肽，以及NGF诱导的PC12细胞向交感神经元的分化是否影响后者的分泌。我们发现如下：1）PC12细胞合成强啡肽原并分泌其肽产物。2）强啡肽原转录本的大小及其主要形式强啡肽的分子量似乎与大鼠垂体前叶中的强啡肽原相似或相同。3）烟碱型胆碱能刺激后，PC12细胞的强啡肽分泌呈剂量依赖性增加，这种效应被特异性尼古丁拮抗剂六甲铵阻断。因此，似乎胆碱能刺激后，PC12细胞的强啡肽与儿茶酚胺共同分泌，这一现象在包括脑啡肽原衍生的阿片肽在内的多种神经肽中都有描述。4）NGF介导的PC12细胞向交感神经元的分化对基础、尼古丁诱导和去极化依赖性的强啡肽分泌产生刺激作用。然而，NGF并未改变强啡肽分泌的尼古丁剂量反应曲线。总之，似乎虽然脑啡肽原衍生肽分泌的变化可能作为PC12细胞向嗜铬细胞分化的标志物，但强啡肽原衍生肽分泌的增加可能代表NGF诱导的PC12细胞向交感神经元分化的标志物。
